Mitochondria-targeted melatonin photorelease supports the presence of melatonin MT1 receptors in mitochondria inhibiting respiration.

The presence of signaling-competent G protein-coupled receptors in intracellular compartments is increasingly recognized. Recently, the presence of Gi/o protein-coupled melatonin MT1 receptors in mitochondria has been revealed, in addition to the plasma membrane. Melatonin is highly cell permeant, activating plasma membrane and mitochondrial receptors equally. Here, we present MCS-1145, a melatonin derivative bearing a triphenylphosphonium cation for specific mitochondrial targeting and a photocleavable o-nitrobenzyl group releasing melatonin upon illumination. MCS-1145 displayed low affinity for MT1 and MT2 but spontaneously accumulated in mitochondria, where it was resistant to washout. Uncaged MCS-1145 and exogenous melatonin recruited ß-arrestin 2 to MT1 in mitochondria and inhibited oxygen consumption in mitochondria isolated from HEK293 cells only when expressing MT1 and from mouse cerebellum of WT mice but not from MT1-knockout mice. Overall, we developed the first mitochondria-targeted photoactivatable melatonin ligand and demonstrate that melatonin inhibits mitochondrial respiration through mitochondrial MT1 receptors.

Retinoic acid receptor α as a novel contributor to adrenal cortex structure and function through interactions with Wnt and Vegfa signalling.

Primary aldosteronism (PA) is the most frequent form of secondary arterial hypertension. Mutations in different genes increase aldosterone production in PA, but additional mechanisms may contribute to increased cell proliferation and aldosterone producing adenoma (APA) development. We performed transcriptome analysis in APA and identified retinoic acid receptor alpha (RARα) signaling as a central molecular network involved in nodule formation. To understand how RARα modulates adrenal structure and function, we explored the adrenal phenotype of male and female Rarα knockout mice. Inactivation of Rarα in mice led to significant structural disorganization of the adrenal cortex in both sexes, with increased adrenal cortex size in female mice and increased cell proliferation in males. Abnormalities of vessel architecture and extracellular matrix were due to decreased Vegfa expression and modifications in extracellular matrix components. On the molecular level, Rarα inactivation leads to inhibition of non-canonical Wnt signaling, without affecting the canonical Wnt pathway nor PKA signaling. Our study suggests that Rarα contributes to the maintenance of normal adrenal cortex structure and cell proliferation, by modulating Wnt signaling. Dysregulation of this interaction may contribute to abnormal cell proliferation, creating a propitious environment for the emergence of specific driver mutations in PA.

Molecular mechanisms in primary aldosteronism

Primary aldosteronism (PA) is the most common form and an under-diagnosed cause of secondary arterial hypertension, accounting for up to 10% of hypertensive cases and associated to increased cardiovascular risk. PA is caused by autonomous overproduction of aldosterone by the adrenal cortex. It is mainly caused by a unilateral aldosterone-producing adenoma (APA) or bilateral adrenal hyperplasia. Excess aldosterone leads to arterial hypertension with suppressed renin, frequently associated to hypokalemia. Mutations in genes coding for ion channels and ATPases have been identified in APA, explaining the pathophysiology of increased aldosterone production. Different inherited genetic abnormalities led to the distinction of four forms of familial hyperaldosteronism (type I to IV) and other genetic defects very likely remain to be identified. Somatic mutations are identified in APA, but also in aldosterone-producing cell clusters (APCCs) in normal adrenals, in image-negative unilateral hyperplasia, in transitional lesions and in APCC from adrenals with bilateral adrenal hyperplasia (BAH). Whether these structures are precursors of APA or whether somatic mutations occur in a proliferative adrenal cortex, is still a matter of debate. This review will summarize our knowledge on the molecular mechanisms responsible for PA and the recent discovery of new genes related to early-onset and familial forms of the disease. We will also address new issues concerning genomic and proteomic changes in adrenals with APA and discuss adrenal pathophysiology in relation to aldosterone-producing structures in the adrenal cortex.

Molecular genetics of Conn adenomas in the era of exome analysis.

Aldosterone-producing adenomas (APA) are a major cause of primary aldosteronism (PA), the most common form of secondary hypertension. Exome analysis of APA has allowed the identification of recurrent somatic mutations in KCNJ5, CACNA1D, ATP1A1, and ATP2B3 in more than 50 % of sporadic cases. These gain of function mutations in ion channels and pumps lead to increased and autonomous aldosterone production. In addition, somatic CTNNB1 mutations have also been identified in APA. The CTNNB1 mutations were also identified in cortisol-producing adenomas and adrenal cancer, but their role in APA development and the mechanisms specifying the hormonal production or the malignant phenotype remain unknown. The role of the somatic mutations in the regulation of aldosterone production is well understood, while the impact of these mutations on cell proliferation remains to be established. Furthermore, the sequence of events leading to APA formation is currently the focus of many studies. There is evidence for a two-hit model where the somatic mutations are second hits occurring in a previously remodeled adrenal cortex. On the other hand, the APA-driver mutations were also identified in aldosterone-producing cell clusters (APCC) in normal adrenals, suggesting that these structures may represent precursors for APA development. As PA due to APA can be cured by surgical removal of the affected adrenal gland, the identification of the underlying genetic abnormalities by novel biomarkers could improve diagnostic and therapeutic approaches of the disease. In this context, recent data on steroid profiling in peripheral venous samples of APA patients and on new drugs capable of inhibiting mutated potassium channels provide promising preliminary data with potential for translation into clinical care.

Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1+ Tim-3+ CD8+ T Cells.

Immune cells are important components of the tumor microenvironment and influence tumor growth and evolution at all stages of carcinogenesis. Notably, it is now well established that the immune infiltrate in human tumors can correlate with prognosis and response to therapy. The analysis of the immune infiltrate in the tumor microenvironment has become a major challenge for the classification of patients and the response to treatment. The co-expression of inhibitory receptors such as Program Cell Death Protein 1 (PD1; also known as CD279), Cytotoxic T Lymphocyte Associated Protein 4 (CTLA-4), T-Cell Immunoglobulin and Mucin Containing Protein-3 (Tim-3; also known as CD366), and Lymphocyte Activation Gene 3 (Lag-3; also known as CD223), is a hallmark of T cell exhaustion. We developed a multiparametric in situ immunofluorescence staining to identify and quantify at the cellular level the co-expression of these inhibitory receptors. On a retrospective series of frozen tissue of renal cell carcinomas (RCC), using a fluorescence multispectral imaging technology coupled with an image analysis software, it was found that co-expression of PD-1 and Tim-3 on tumor infiltrating CD8+ T cells is correlated with a poor prognosis in RCC. To our knowledge, this represents the first study demonstrating that this automated multiplex in situ technology may have some clinical relevance.

A gain-of-function mutation in the CLCN2 chloride channel gene causes primary aldosteronism.

Primary aldosteronism is the most common and curable form of secondary arterial hypertension. We performed whole-exome sequencing in patients with early-onset primary aldosteronism and identified a de novo heterozygous c.71G>A/p.Gly24Asp mutation in the CLCN2 gene, encoding the voltage-gated ClC-2 chloride channel 1 , in a patient diagnosed at 9 years of age. Patch-clamp analysis of glomerulosa cells of mouse adrenal gland slices showed hyperpolarization-activated Cl- currents that were abolished in Clcn2-/- mice. The p.Gly24Asp variant, located in a well-conserved 'inactivation domain'2,3, abolished the voltage- and time-dependent gating of ClC-2 and strongly increased Cl- conductance at resting potentials. Expression of ClC-2Asp24 in adrenocortical cells increased expression of aldosterone synthase and aldosterone production. Our data indicate that CLCN2 mutations cause primary aldosteronism. They highlight the important role of chloride in aldosterone biosynthesis and identify ClC-2 as the foremost chloride conductor of resting glomerulosa cells.